NDLI: Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

Content Provider	PubMed Central
Author	Valkonen, Mari Dominik, Mojzita Penttilä, Merja Benčina, Mojca
Copyright Year	2013
Abstract	The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.
Related Links	http://dx.doi.org/10.1128/aem.02515-13
Ending Page	7187
Page Count	9
Starting Page	7179
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	23
Volume Number	79
Language	English
Publisher	American Society for Microbiology
Publisher Date	2013-12-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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